2
Introduction Rotavirus is the most common cause of severe diarrhea in infants and young children worldwide. Globally it is responsible for 611,000 childhood deaths [1], and another 2 million hospitalizations [2] every year. More than 80% deaths occur in low-income countries [1], and India records the highest mortality [3]. Rotavirus diarrhea accounts for 2,000,000 outpatient visits, 457, ,000 hospitalizations, and 122, ,000 deaths in under-5 children in India annually. There is huge economic impact of rotavirus diarrhea. It is estimated that India spends approximately Rs 1.8–3.2 billion (US$ 37.4 to 66.8 million) in direct medical costs annually and Rs 107–176 million (US$ 2.2–3.7 million) in direct non-medical costs for the treatment of rotavirus diarrhea in children <5 years of age, with a total burden of Rs 2.0–3.4 billion (US$ 41–72 million) [4].

3
Epidemiology Rotaviruses are double stranded RNA viruses comprising a genus within the family Reoviridae. The primary mode of transmission is via the fecal- oral route with symptoms typically developing after an incubation period of one to two days. Majority of children become infected with rotavirus within the first three years of life, with a peak incidence of rotavirus diarrhea between six to 24 months of age [5,6].

5
Molecular epidemiology The mature virus particles are triple layered, with an approximate diameter of 70 nm and icosahedral symmetry. The rotavirus genome consists of 11 segments of double-stranded RNA, which code for 6 structural viral proteins, VP1, VP2, VP3, VP4, VP6 and VP7 and 6 non-structural proteins, NSP1-NSP6 [20], where gene segment 11 encodes both NSP5 and 6. Genome is encompassed by an inner core consisting of VP2, VP1 and VP3 proteins. Intermediate layer or inner capsid is made of VP6 determining group and subgroup specificities. The outer capsid layer is composed of two proteins, VP7 and VP4 eliciting neutralizing antibody responses. Rotaviruses are classified into seven different groups (A-G) which is based on the antigenic specificity of the VP6 capsid proteins, as well as on the pattern of electrophoretic mobility of the 11 RNA segments of the viral genome.

6
Molecular epidemiology Groups A, B and C are known to infect humans. Severe, life- threatening disease in children worldwide is caused predominantly by group A rotaviruses. Within group A, four different subgroups (SG); SGI, SGII, SGI and II, and nonI/nonII, have been distinguished on the basis of VP6 diversity, among which possibly SGI or SGII are the only humans strains [21]. Further typing schemes to describe rotavirus strains are based on the proteins of the outer capsid eliciting neutralizing antibodies, VP7 (G serotypes) and VP4 (P serotypes). G and P serotypes are defined on the basis of their reactivity to specific monoclonal antibodies. Variability in the genes encoding the two outer capsid proteins VP7 and VP4 form the basis of the current strain typing of group A rotaviruses into G and P genotypes. All known G serotypes correspond with genotypes; though, more P genotypes than serotypes have been identified.

7
Molecular epidemiology Current practice is to identify a rotavirus strain by a G genotype, indicated by a number, followed by its P type. To distinguish strains identified by P genotyping from those identified by P serotyping, the dual serotype/genotype nomenclature is used. P genotypes are expressed as P followed by a number in square brackets whereas P serotypes are designated as P with serotype number, followed by corresponding genotype in square brackets (e.g., the same strain could be represented as G9P[6], when both G and P genotypes are used, or G9P2A[6] when G genotype is followed by P serotype/genotype classification). Till date, at least 15 G genotypes and 25 P genotypes have been identified [22].

8
Nosocomial diarrhea The Centers of Disease Control and Prevention defines nosocomial diarrhea as an acute onset of diarrhea in hospitalized patient, characterized by liquid stool for more than 12 hr with or without vomiting and/or fever or the occurrence of symptoms like nausea, vomiting, abdominal pain in conjunction with objective evidence of enteric infection obtained by stool culture, antigen or antibody assay of feces or blood, routine microscopic examination of stool or toxin assay. A period of at least 3 days of hospitalization prior to the onset of diarrhea is necessary to term it as nosocomial diarrhea [23].

9
Objective The high burden of rotavirus disease in India requires that there is an urgent need to summarize and critically evaluate all available information related to the prevalence of rotavirus diarrhea in India. Comprehensive national estimates of the burden of diarrheal diseases caused by rotavirus in India are still lacking. The objective of this systematic review is to estimate the burden of rotavirus diarrhea in the community and in hospital in children <5 years of age in India.

10
Methods Study Design We reviewed the epidemiology of rotavirus diarrhea in under-5 Indian children. The standard methodology for conducting a narrative systematic review was used [12]. Our primary research question was ‘epidemiology of rotavirus diarrhea in under-5 children of India’.

11
Secondary research questions (1)Epidemiology of rotavirus diarrhea in under-5 hospitalized children from different parts of India (2) Epidemiology of rotavirus diarrhea in under-5 children in community from different parts of India (3) Demographic profile of rotavirus diarrhea in under-5 Indian children (4) Identification of molecular epidemiology of rotavirus diarrhea in under-5 children from different parts of India (5) Incidence of rotavirus infection as a cause of nosocomial diarrhea in under-5 Indian children, and (6) Mortality from rotavirus diarrhea in under-5 Indian children

12
The primary databases searched were Medline through PubMed (www.pubmed.com) and IndMed (http://indmed.nic.in/). Other sources including World Health Organization (WHO) reports available online (www.who.int), documents of the UNICEF available online (www.unicef.org/india/), National Family Health Survey (http://www.nfhsindia.org/), and documents of the Ministry of Health and Family Welfare, Government of India (www.mohfw.nic.in) available online were also accessed. We also searched the related articles and the reference lists of included publications to identify additional studies.

13
Inclusion and Exclusion Criteria We included all types of publications available in scientific public domain and reporting on rotavirus infection in India by direct data collection through clinical examination, and/or laboratory testing. No particular time frame was set. We limited our search in humans, and articles published in English language. Participants Children: 1 month to 5 years.

14
Searching the Literature For searching the PubMed, a search string was devised by converting each research question into problem, intervention, comparison, outcome (PICO) format. MeSH headings were looked for the research theme in question and added to the PubMed search builder. Salient keywords were included during search. A search for MeSH headings for ‘rotavirus’, revealed ‘Rotavirus, infections’, which was relevant and yielded 31 subheadings from which we selected ‘rotavirus diarrhea’, ‘rotavirus gastroenteritis’, and ‘rotavirus disease’.

15
Searching the Literature For assessing the epidemiology of rotavirus diarrhea in India, we searched PubMed using the search string: “(epidemiology* OR burden OR morbidity OR mortality or incidence OR prevalence OR profile) AND (rotavirus OR rotavirus diarrhea OR rotavirus gastroenteritis OR rotavirus disease) AND India. An additional search was made for the secondary research questions by combining keywords/MeSH terms for the secondary research question using the search string “(*) AND (rotavirus, infections) AND India”, where the asterisk represents the MeSH term/keywords for the secondary research question.

16
Searching the Literature To search the IndMed, the search string was kept simple using search keywords. The search date, search terms, search string and search output were recorded and saved. In the next step, we scrutinized all titles and excluding the titles which were obviously not relevant; the remaining articles were studied further.

17
Searching the Literature The next step involved examination of the abstract or the introduction (where the abstract was not published) of the short-listed titles; the ones which were not found relevant were excluded and the remaining articles were processed further. In the next step examination of full-text articles were done. Related articles and cross-references in identified articles were also reviewed and similar steps were performed before short listing the cross-references.

20
Burden of Rotavirus diarrhea in hospitalized children from different parts of India A total of 49 studies [24-72], carried out using samples obtained from under-5 Indian children hospitalized with rotavirus diarrhea were included (Table II, Fig. 1A, Fig. 1B). The studies were conducted in various geographic locations. Eighteen studies were carried in Northern India, 7 in Western India, 8 in Eastern India and 11 in Southern India. Six studies involved multiple geographical locations. Most of the studies used ELISA/PAGE for the screening of rotavirus although latex agglutination assay, immunoblot and electron microscopy were also used in a limited number of studies.

21
Burden of Rotavirus diarrhea in hospitalized children from different parts of India A total of stool samples were tested, of which 7448 (21.7%) tested positive. Rates of rotavirus positivity ranged from 4.6% in Kolkata, Ghosh, et al.[51] to 89.8% in Manipur, Sengupta, et al.[54]. Largest number of samples came from Northern India (9430), followed by Southern India (8840), multicentric studies (7428), Western India (5927) and Eastern India (2782). Region-wise, highest positivity was recoded from Eastern India (31.7%), followed by Western India (20.8%), Northern India (17.4%) and Southern India (17.2%). Studies conducted in multiple locations recorded an incidence of 21.4%, replicating the overall incidence of rotavirus diarrhea in hospitalized children in India. Details of studies have been summarized in Web Table I.

22
TABLE II: Rotavirus Positivity in Children Hospitalized with Diarrhea from Different Parts of India

23
TABLE II: Rotavirus Positivity in Children Hospitalized with Diarrhea from Different Parts of India contd…

28
Figure 1A: Overall Rotavirus positivity in Indian children hospitalized with diarrhea Figure 1B: Region-wise breakup of Rotavirus positivity in children hospitalized with diarrhea

29
Burden of Rotavirus diarrhea in the community from different parts of India A total of 15 studies, carried out using samples obtained from under-5 Indian children in community with rotavirus diarrhea were included (Table III, Fig. 2A, 2B). Nine studies were carried in Northern India, 2 in Western India, 1 in Eastern India and 3 in Southern India. A total of 7566 stool samples were tested in children with diarrhea and 2810 controls, 1504 (19.9%) were tested positive in children with diarrhea and 117 (4.2%) in controls. Rates of rotavirus positivity in diarrhea ranged from 33.7% in Manipur [83], to 4% in Delhi, [74]. In controls, stool positivity ranged from 0 in Vellore [84] to 12.3% in North India [76]. Details of studies have been summarized in Web Table II.

30
TABLE III: Burden of Rotavirus Diarrhea in the Community from Different Parts of India

31
TABLE III: Burden of Rotavirus Diarrhea in the Community from Different Parts of India contd..

32
Figure 2A: Overall Rotavirus positivity in Indian children from community Figure 2B: Region-wise breakup of Rotavirus positivity in children with and without diarrhea from community

33
Demographic profile of rotavirus diarrhea Demographic profile of rotavirus diarrhea has been summarized in Table IV and Web Table III. A total of 35 studies could be identified which described demographic profile of rotavirus diarrhea in children. Details of all the studies have been discussed in Web Table III, 27 have been summarized in Table IV. Most cases of rotavirus diarrhea were found to occur in the first two years of life, most commonly affected age group was 7-12 months both in hospital and community settings. Most of the authors have observed an increase in rotavirus-associated diarrhea during the winter months.

36
Rotavirus serotypes detected from various parts of India A total of 46 studies could be identified which dealt with serotyping of rotavirus, of which G and P typing were done in 40 studies. Details of studies have been summarized in Table V, VI, VII, Figure and Web Table IV. Maximum number of studies was reported from southern India (12), 8 from western India, 7 from eastern India and 6 studies were reported from northern India. Seven studies involved multiple locations.

37
Rotavirus G serotypes detected from various parts of India Overall, G1 was the most common serotype isolated in Indian studies (32%), followed by G2 (24%) and G- untypable (15%). On regional distribution, in northern India, G1 was the most common serotype isolated (32%), followed by G2 (20%), G-untypable (13%) and G9(11%). In western India, G2 was predominant (28%), closely followed by G-untypable (25%) and G1 (23%). In eastern India, G1 was detected in 40%, followed by G2 (30%). In southern India, G1 was predominant (31%), followed by G2 (23%) and G-untypable (21%).

38
Figure 3

39
Figure 4-7: G serotyping

40
Rotavirus P serotypes detected from various parts of India In P serotyping, P[4] was most prevalent (23%) all over India, followed by P[6] (20%) and P Untypable/others (13%). In northern India, P[6] was most common (22%), followed by P[4] (19%). Prevalence of P[4] was 43% in western India and 32% in eastern India. In southern India, untypable P type was documented in 40%, followed by P[8] (39%). P[4] was documented in 17%.

41
Figure 8

42
Figure 9-12: P serotyping

43
Nosocomial Rotavirus Infections Five studies were identified which dealt with nosocomial rotavirus infection. Two studies were done in Kolkata [ ], one in Delhi [110], two in Vellore [113,114]. A total of 3810 children hospitalized for illnesses other than diarrhea were included. Among them 466 (12.2%) developed nosocomial diarrhea. Rotavirus was identified as the etiological agent in 75 (16.1%) of them. Details of the studies have been discussed in Table VIII, Figure 13 and Web Table V.

44
Figure 13: Nosocomial Rotavirus Infections Five studies were included in nosocomial rotavirus infection. One study was done in Delhi [110], two studies in Kolkata [ ] and two in Vellore [113,114].

45
Rotavirus mortality Indian Rotavirus Strain Surveillance Network (IRSSN) data, which covered pediatric admissions to 10 hospitals in 7 cities of India from December 2005 to November 2007, showed that stool samples from 1405 (39%) of 3580 children hospitalized with diarrhea were positive for rotavirus [72]. The Million Death Study, a nationally representative sample of 6.3 million people in 1.1 million households within the Sample Registration System, recorded approximately diarrheal deaths in India during 2005, i.e., 1 in 82 Indian children died from diarrhea before the age of 5 years [128]. As per the IRSSN data, rotavirus was estimated to cause approximately 34% (113,000; 99% confidence interval, CI: 86,000–155,000) of all diarrheal deaths in under-5 children.

46
Rotavirus mortality As per the IRSSN data, rotavirus was estimated to cause approximately 34% (113,000; 99% confidence interval, CI: 86,000–155,000) of all diarrheal deaths in under-5 children. Taken together, there was an estimated mortality rate of 4.14 (99% CI: 3.14–5.68) deaths per 1000 live births during 2005 suggesting that approximately 1 in 242 children will die from rotavirus infection before reaching their fifth birthday. Rotavirus-associated mortality rate varied by gender, age and region during 2005 [129]. The rate among girls (4.89 deaths per 1000 live births; 99% CI: 3.75–6.79) was 42% higher than among boys (3.45 deaths per 1000 live births; 99% CI: 2.58–4.66).

47
Rotavirus mortality Region-wise, most rotavirus-associated deaths during 2005 occurred in the central India and eastern India (56,400 vs. 28,900, respectively). State-wise mortality rate per 1000 live births due to rotavirus infection among Indian under-5 children during 2005 is shown in Fig. 14. The mortality rate from rota­virus associated gastroenteritis among under-5 children ranged from a low of 1.64 deaths per 1000 live births in western India to as high as 5.49 deaths per 1000 live births in central India. Overall mortality rate was 4.1 per 1000 live births.

48
Rotavirus mortality Highest mortality rate was observed in Bihar (6.3) followed by Uttar Pradesh (6.2). Lowest rate was recorded by Maharashtra (1.1). In absolute figures, more than half (64,400) of deaths were estimated to occur in three states, Uttar Pradesh recording the highest number (35,700), followed by Bihar (17,800) and Madhya Pradesh (10,900) [127]. The age distribution of deaths from rotavirus infection was similar for boys across the country, but age of death was lower in girls in the northern and southern India compared to other regions.

49
Figure 14: State-wise estimated mortality rate due to rotavirus infection among Indian under-5 children during 2005

50
Discussion Limitations Lack of data from many geographical locations, particularly central India, where the burden of diarrheal disease is particularly heavy. There are many gaps in our understanding of the epidemiology of rotavirus diarrhea in under-5 children particularly at the community level. Most of the studies were confined to few places in each region except southern India and sample size differed widely in individual studies. Another shortcoming is use of different methodologies employed for virus detection. The limitation is particularly applicable to the period prior to 1994 when molecular methods for serotyping were not available. Lastly, we did not perform any formal quality assessment of the individual studies beyond selection criteria requirements.

51
Discussion The burden of rotavirus diarrhea is high in India (GRADE quality of evidence moderate) Incidence of rotavirus positive diarrhea is higher in hospitalized children than in the community settings. Rotavirus infection is most common below 2 years of age, 7-12 months being the most commonly affected age group. Maximum infection occurs in winter months, from October– February. Mortality from rotavirus is still very high in India. There are large regional differences in the pattern of circulating rotavirus strains. Overall, G1 was the most common serotype isolated in Indian studies (32%), followed by G2 (24%) and G- untypable (15%).

52
Discussion Beyond the common G1, G2, G3 and G4, more unusual strains like G9 and G12 are appearing as causative agents. Mixed infections, along with human-animal reassortments, unusual G-types (G6 and G8) and strains (G3P[11] and G9P[10]) had been described which highlights the wide genetic and antigenic diversity of strains circulating in different regions. Even group B and group C rotavirus have been incriminated to cause diarrhea. Such variation necessitates continuous surveillance. In view of continuing high morbidity and mortality from rotavirus diarrhea and enormous economic consequences thereof, there is a strong case for immunization against rotavirus infection. It is also crucial to assess and consider the strain variability in the design of a new candidate vaccines and its clinical evaluation in regions with high strain diversity.

53
Discussion Also after introduction of immunization, it is important to have ongoing surveillance to measure the impact of large- scale vaccination program on rotavirus diarrhea hospitalization and mortality rates. As per latest WHO estimate, India as a country records highest rotavirus deaths in under-5 children (22%). Unfortunately, very few studies dealt with mortality from rotavirus diarrhea. Particularly, there is no study from Bihar and very few studies in Uttar Pradesh, the states which record highest mortality rate from rotavirus diarrhea. More studies are needed at the community level to better define the epidemiology of rotavirus diarrhea.

54
Conclusions Rotavirus diarrhea is a significant public health problem in India (GRADE quality of evidence moderate). The incidence of rotavirus positive diarrhea is higher in hospitalized children than in community, indicating thereby that rotavirus accounts for more severe dehydrating diarrhea in children. Most cases of rotavirus diarrhea in India are caused by G1, G2, and G untypable strains with distinct regional variations. Rotavirus is also an important cause of nosocomial diarrhea in children. In view of continuing high morbidity and mortality from rotavirus diarrhea and enormous economic consequences thereof, there is a strong case for immunization against rotavirus infection in India.